The yeast Pichia pastoris has previously is a suitable production host for the manufacture of recombinant proteins of therapeutic utility. Examples include the production of Human Serum Albumin and the Kallikrein inhibitor, Ecallantide (Reichert, J. mAbs 4:3 1-3, 2012). Pichia pastoris has been used for the production of recombinant monoclonal antibodies having correctly assembled heavy and light chains (U.S. Pat. No. 7,927,863). The glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter can drive expression in yeast of an antibody lacking N-glycosylation in yeast (U.S. Pat. No. 7,927,863). Recent work by Baumann et. al. (BMC Genomics 2011, 12:218), using the GAP system to produce recombinant antibody Fab fragment has shown that this system, previously thought to be constitutive, exhibits increased expression under hypoxic conditions using glucose as the source of carbon and energy. Hypoxic conditions are those that allow the dissolved oxygen level in a fermentation to drop to very low levels while still supplying oxygen to the culture through aeration and agitation. This results in mixed aerobic and fermentative metabolism.
The use of hypoxic conditions in a fermentor can result in the toxic accumulation of ethanol, and care must be exercised to control the process such that toxic levels do not accumulate. Baumann accomplished this by measuring the level of ethanol in the fermentor and adjusting the glucose feed rate to reduce its accumulation. However this method is not very scalable, as technology for reliably measuring ethanol in large scale fermentors is not widely available.
There is a continuing need in the art for efficient and accurate production of recombinant proteins, especially antibody molecules, antibody fragments, and antibody constructs.